Characteristics of the interaction of calcium with casein submicelles as determined by analytical affinity chromatography.
Interaction of calcium with casein submicelles was investigated in CaCl2 and calcium phosphate buffers and with synthetic milk salt solutions using the technique of analytical affinity chromatography. Micelles that had been prepared by size exclusion chromatography with glycerolpropyl controlled-pore glass from fresh raw skim milk that had never been cooled, were dialyzed at room temperature against calcium-free imidazole buffer, pH 6.7. Resulting submicelles were covalently immobilized on succinamidopropyl controlled-pore glass (300-nm pore size). Using 45Ca to monitor the elution retardation, the affinity of free Ca2+ and calcium salt species was determined at temperatures of 20 to 40 degrees C and pH 6.0 to 7.5. Increasing the pH in this range or increasing the temperature strengthened the binding of calcium to submicelles, similar to previous observations with individual caseins. However, the enthalpy change obtained from the temperature dependence was considerably greater than that reported for alpha s1- and beta-caseins. Furthermore, the elution profiles for 45Ca in milk salt solutions were decidedly different from those in CaCl2 or calcium phosphate buffers and the affinities were also greater. For example, at pH 6.7 and 30 degrees C the average dissociation constant for the submicelle-calcium complex is 0.074 mM for CaCl2 and calcium phosphate buffers, vs 0.016 mM for the milk salt solution. The asymmetric frontal boundaries and higher average affinities observed with milk salts may be due to binding of calcium salts with greater affinity in addition to the binding of free Ca2+ in these solutions.
Jang
H D
HD
Department of Food Science, Southeast Dairy Foods Center, North Carolina State University.
Swaisgood
H E
HE
D000818:Animals; D002118:Calcium / Q000378:metabolism*; D002132:Calcium Radioisotopes; D002364:Caseins / Q000302:isolation & purification / Q000378:metabolism*; D002417:Cattle; D002846:Chromatography, Affinity; D007700:Kinetics; D008297:Male; D008823:Micelles; D008892:Milk; D008962:Models, Theoretical; D013816:Thermodynamics
D002132:Calcium Radioisotopes; D002364:Caseins; D008823:Micelles; D002118:Calcium